Method for quantifying cholesterol in high density lipoprotein

ABSTRACT

Disclosed is a method for quantifying cholesterol in HDL, which does not require complex fragmentation and separation operations, and by which the HDL cholesterol in test samples containing HDL and other lipoproteins such as low density lipoprotein (LDL), very low density lipoprotein (VLDL) and chylomicron (CM) may be quantified selectively, simply and accurately. The method for quantifying cholesterol in high density lipoprotein comprises a first step of erasing cholesterol in lipoproteins other than high density lipoprotein in a test sample, and a second step of adding a surfactant which specifically acts on high density lipoprotein to the product of the first step and enzymatically quantifying cholesterol in high density lipoprotein.

This application is a divisional of co-pending application Ser. No.09/117,806, filed on Aug. 6, 1998 and for which priority is claimedunder 35 U.S.C. § 120. application Ser. No. 09/117,806 is the nationalphase of PCT International Application No. PCT/JP97/04442 filed on Dec.4, 1997 under 35 U.S.C. § 371. The entire contents of each of theabove-identified applications are hereby incorporated by reference. Thisapplication also claims priority of Application No. 344649/96 tiled inJapan on Dec. 9, 1996 under 35 U.S.C. § 119.

TECHNICAL FIELD

The present invention relates to a method for quantifying cholesterol inhigh density lipoprotein (hereinafter also referred to as “HDL”)

BACKGROUND ART

It is known that HDL relates to removal of cholesterol accumulated incells because it receives cholesterol from various tissues includingwalls of blood vessels with arterial sclerosis, so that HDL is usefulfor estimating the risk for various arterial sclerosises includingcoronary artery sclerosis, and that its blood level is an indicator forthe risk of onset of arterial sclerosis.

Methods for measuring cholesterol in HDL include a method in which HDLis separated from other lipoproteins by ultracentrifugation and then theHDL is measured; and a method in which the cholesterol in HDL isseparated by electrophoresis, then the lipid is stained, and theintensity of the generated color is measured. However, these methods arecomplex or a number of samples cannot be assayed, so that they are notcommonly used.

The method for measuring the cholesterol in HDL, which is generally usedin the field of clinical test is the method in which a precipitatingagent is added to the sample so as to coagulate the lipoproteins otherthan HDL, removing the coagulated lipoproteins by centrifugation, andthe cholesterol in the resulting supernatant containing HDL alone ismeasured. Although this method is simpler than the ultracentrifugationmethod and the electrophoresis method, it is not satisfactorily simplebecause it comprises addition of the precipitating agent and subsequentseparation, and a comparative large amount of sample is needed.

On the other hand, methods in which the cholesterol in HDL is separatelyquantified by using enzymes have been proposed. For example, a method isknown, which comprises the steps of preliminarily coagulating thelipoproteins other than HDL by an antibody and polyanion, enzymaticallyreacting the cholesterol in HDL alone, inactivating the enzyme andsimultaneously re-dissolving the coagulated mass, and measuring theabsorbance of the resulting solution (Japanese Laid-open PatentApplication (Kokai) No. 6-242110). However, this method has a problem inthat it is necessary to add reagents at least three times, so that thismethod can be practiced only by the limited analyzing apparatuses.Therefore, this method is not widely used.

Other methods include a method in which an enzyme reaction is carriedout in the presence of a bile salt or a nonionic surfactant (JapaneseLaid-open Patent Application (Kokai) No. 63-126498); a more recentlydeveloped method in which the cholesterol in HDL is specifically trappedby chemically modified cholesterol esterase and/or cholesterol oxidasein the presence of a clathrate compound such as cyclodextrin (JapaneseLaid-open Patent Application (Kokai) No. 7-301636); and a method inwhich the lipoproteins other than HDL are made into aggregates orcomplexes and then the cholesterol in HDL is trapped by an enzymereaction (Japanese Laid-open Patent Application (Kokai) Nos. 8-131197and 8-201393). However, with these methods, the results for certainsamples are different from the results by the precipitation method, sothat their specificities are problematic.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a method forquantifying cholesterol in HDL, which does not require complexfragmentation and separation operations, and by which the HDLcholesterol in test samples containing HDL and other lipoproteins suchas low density lipoprotein (LDL), very low density lipoprotein (VLDL)and chylomicron (CM) may be quantified selectively, simply andaccurately.

The present inventors intensively studied to discover that surfactantswhich act on HDL but substantially do not act on other lipoproteinsexist. The present inventors further discovered that HDL cholesterol intest samples containing HDL and other lipoproteins may be selectively,simply and accurately quantified by selectively erasing the cholesterolin the lipoproteins other than the high density lipoprotein in the testsample and then enzymatically quantifying the cholesterol originatedfrom HDL in the presence of the above-mentioned surfactant, therebycompleting the present invention.

That is, the present invention provides a method for quantifyingcholesterol in high density lipoprotein, comprising a first step oferasing cholesterol in lipoproteins other than high density lipoproteinin a test sample, and a second step of adding a surfactant whichspecifically acts on high density lipoprotein to the product of thefirst step and enzymatically quantifying cholesterol in high densitylipoprotein.

By the method of the present invention, the cholesterol in HDL in a testsample containing HDL and other lipoproteins such as LDL, VLDL and CMmay be quantified selectively, simply and accurately, without complexfragmentation and separation operations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows correlation between the amounts of the cholesterol in HDLmeasured by the method of an example according to the present inventionand the amounts of the cholesterol in HDL measured by the conventionalprecipitation method.

FIG. 2 shows correlation between the amounts of the cholesterol in HDLmeasured by the method of another example according to the presentinvention and the amounts of the cholesterol in HDL measured by theconventional precipitation method.

FIG. 3 shows correlation between the amounts of the cholesterol in HDLmeasured by the method of a still another example according to thepresent invention and the amounts of the cholesterol in HDL measured bythe conventional precipitation method.

BEST MODE FOR CARRYING OUT THE INVENTION

Cholesterols contained in lipoproteins include ester type cholesterol(cholesterol ester) and free cholesterol. In this specification, theterm “cholesterol” includes both of these unless otherwise specified.

The test sample subjected to the method of the present invention may beany sample which may contain lipoproteins such as HDL, LDL, VLDL and CM.Examples of the test samples include body fluids such as sera as well asdilutions thereof, although the test samples are not restricted thereto.

In the first step in the method of the present invention, thecholesterol in the lipoproteins other than HDL in the test sample isselectively erased. The term “erase” herein means to decompose thecholesterol and to make the decomposed products undetectable in thesubsequent second step. The methods for selectively erasing thecholesterol in the lipoproteins other than HDL, that is, in LDL, VLDL,CM and the like include the following methods.

In the first method, cholesterol esterase and cholesterol oxidase areacted on the test sample in the absence of a surfactant which acts onHDL, and the generated hydrogen peroxide is removed. By the action ofcholesterol esterase, the ester type cholesterol in the lipoproteins arehydrolyzed to yield free cholesterol and fatty acids. The thus generatedfree cholesterol and the free cholesterol inherently existing in thelipoproteins are oxidized by the action of cholesterol oxidase to yieldcholestenone and hydrogen peroxide. The thus generated hydrogen peroxideis removed. Methods for removing hydrogen peroxide include a method inwhich the hydrogen peroxide is decomposed to water and oxygen bycatalase; and a method in which a phenol-based or aniline-based hydrogendonor compound, such as DAOS(N-ethyl-N-(2-hydroxysulfopropyl)-3,5-dimethoxyaniline), which reactswith hydrogen peroxide to yield a colorless quinone, is reacted with thehydrogen peroxide to convert the hydrogen peroxide to the colorlessquinone, although the methods for removing hydrogen peroxide are notrestricted to these methods.

In the above-mentioned first step, by carrying out the step in theabsence of a surfactant which acts on HDL, the cholesterol in HDL is notsubstantially reacted, while the cholesterol in the other lipoproteinssuch as LDL, VLDL and CM are reacted and erased. By this, in thesubsequent second step, the cholesterol in HDL is selectivelyquantified.

The concentration of the cholesterol esterase in the reaction mixture inthe first step may preferably be about 0.2 to 1.0 U/ml, and theconcentration of the cholesterol oxidase may preferably be about 0.1 to0.7 U/ml. The concentration of the catalase may preferably be about 40to 100 U/ml and the concentration of the peroxidase may preferably beabout 0.4 to 1.0 U/ml. The concentration of the compound which yieldsthe colorless quinone upon reaction with hydrogen peroxide maypreferably be about 0.4 to 0.8 mmol/l.

The reaction in the first step may preferably be carried out in a bufferwith a pH of 5 to 8, and the buffer may preferably be phosphate buffer,glycine buffer, Tris buffer or Good's buffer. Especially, Bis-Tris,PIPES, MOPSO, BES, HEPES and POPSO which are Good's buffer arepreferred. The concentration of the buffer may preferably be about 10 to500 mM.

To increase the degree of erasing of the lipoproteins other than HDL,divalent metal ion may be contained in the reaction mixture. Preferredexamples of the divalent metal ion include copper ion, iron ion andmagnesium ion. Among these, magnesium ion is especially preferred. Theconcentration of the divalent metal ion may preferably be about 5 to 200mM.

A lipoprotein hydrolase may optionally be added to the reaction mixturein the first step. Addition of this enzyme is preferred becauseespecially the cholesterol in VLDL easily reacts. The concentration ofthis enzyme in the reaction mixture may preferably be about 5.0 to 10.0U/ml.

The reaction temperature in the first step may preferably be about 25°C. to 40° C., and 37° C. is best preferred. The reaction time may beabout 2 to 10 minutes.

In the following second step, a surfactant which specifically acts onHDL is added to the reaction product of the first step, and thecholesterol in high density lipoprotein is enzymatically quantified. Theterm “surfactant which specifically acts on HDL” means a surfactant bywhich the cholesterol in HDL reacts due to the action of an enzyme suchas cholesterol esterase or cholesterol oxidase (the reaction ratio isnot less than 70%, preferably not less than 90%), while the cholesterolin the lipoproteins other than HDL does not substantially react (thereaction ratio is not more than 30%, preferably not more than 20%).Examples of such a surfactant include the surfactants having ahydrophilicity lipophilicity balance (HLB) of 13 to 14, especiallynonionic surfactants with a HLB of 13 to 14, especially polyalkyleneoxide derivatives. Preferred examples of the derivatives here includecondensation products with higher alcohols, condensation products withhigher fatty acids, condensation products with higher fatty acid amides,condensation products with higher alkylamines, condensation productswith higher alkylmercaptane and condensation products with alkylphenols. Among the polyalkyleneoxide derivatives, polyethylene oxidederivatives are best preferred. The above-mentioned range of HLB may beattained by mixing a plurality of surfactants, and such a mixture of aplurality of surfactants may also be used. The method for calculatingHLB of surfactants is well-known, and is described in, for example,Hiroshi HORIGUCHI, “New Surfactants”, 1986, Sankyo Shuppan.

Preferred specific examples of the surfactant include polyoxyethylenelauryl ether, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether,polyoxyethylene higher alcohol (C₄-C₃₅) ether, polyoxyethylene octylphenyl ether, polyoxyethylene nonylphenyl ether and the like, althoughthe surfactant is not restricted thereto.

Although the concentration of the surfactant in the second step is notrestricted, it may preferably be 0.05 to 3% by weight, more preferably0.1 to 1.5% by weight based on the total reaction mixture.

In the presence of the above-mentioned surfactant, the HDL cholesterolin the test sample may be enzymatically quantified. That is, in thefirst step, most of the cholesterol in the lipoproteins other than HDLis erased, and with the synergistic effect with the reaction in thesecond step, the cholesterol in HDL alone is quantified.

The method for enzymatically quantify cholesterol per se is well-knownin the art. For example, as in the first step, cholesterol may bequantified by generating hydrogen peroxide from cholesterol ester andfree cholesterol by the action of cholesterol esterase and cholesteroloxidase, and by quantifying the generated hydrogen peroxide.Quantification of hydrogen peroxide may be carried out by, for example,reacting the hydrogen peroxide with a compound which forms a quinonepigment, and by measuring the amount of the generated quinone pigment bymeasuring absorbance or the like. The quinone pigment may be formed by,for example, reacting hydrogen peroxide and 4-aminoantipyrine and DAOSor HDAOS (N-(2-hydroxysulfopropyl)-3,5-dimethyoxyaniline). The quinonepigment formed thereby has the maximum absorbance at 593 nm when DAOS isused, and has the maximum absorbance at 583 nm when HDAOS is used.Although the concentration of the compound which yields the quinonepigment is not restricted, the concentration of 4-aminoantipyrine, forexample, may preferably be 0.1 to 2.0 mM, more preferably 0.5 to 1.5 mM,and the concentration of DAOS or HDAOS may preferably be 0.1 to 1.5 mM,more preferably 0.4 to 1.0 mM. Although the concentration of theperoxidase is not restricted, it may preferably be 0.4 to 5 U/ml in thetotal reaction mixture. Preferred reaction conditions (reactiontemperature, reaction time, buffer and pH) are the same as the preferredreaction conditions in the first step.

In cases where the generated hydrogen peroxide is decomposed withcatalase, a catalase inhibitor such as sodium azide is used in thesecond step so as to inhibit the catalase because it is necessary toinhibit the catalase in the second step.

EXAMPLES

The present invention will now be described more concretely by way ofexamples thereof. It should be noted, however, the present invention isnot restricted to the examples below. In the examples below, all “%” areby weight unless otherwise specified.

Reference Example 1

Using samples containing known amounts of purified HDL, LDL, VLDL andCM, respectively, the cholesterol in each of the lipoproteins wasenzymatically quantified in the presence of a nonionic surfactantEmulgen 911 (polyoxyethylene nonyl ether, HLB 13.7), Emulgen B66(polyoxyethylene derivative, HLB 13.2) or a mixture of Emulgen B66 andEmulgen A90 (polyoxyethylene derivative, HLB 14.5), all of which arecommercially available from KAO CORPORATION. This operation was carriedout as follows.

To a solution containing 0.5 U/ml cholesterol esterase, 0.4 U/mlcholesterol oxidase, 0.5 U/ml peroxidase, 1.0 mmol/l 4-aminoantipyrineand 0.5 mmol/l HDAOS in 50 mM PIPES buffer, pH 7.0, Emulgen 911 orEmulgen B66 was added to a concentration of 0.1% by weight, or EmulgenB66/Emulgen A90 mixture (9/1) was added to a concentration of 1.3% byweight. Twenty microliters of each sample was mixed with 2.0 ml of thethus prepared mixture and the resulting mixture was allowed to react at37° C. for 10 minutes, followed by measuring absorbance at 600 nm.

As a result, the reaction ratio (i.e., the ratio of the quantifiedcholesterol in the total cholesterol) was about 95% for the cholesterolin HDL, and about 18 to 22% for the cholesterols in other lipoproteins.

From this, it can be seen that Emulgen 911, Emulgen B66 and the EmulgenB66/Emulgen A90 mixture are within the scope of the term “surfactantwhich specifically acts on high density lipoprotein”.

Example 1

First reagents and second reagents having the following compositions,respectively, were prepared.

First Reagents PIPES buffer, pH 7.0 100 mmol/l HDAOS 0.7 mmol/lCholesterol esterase 0.8 U/ml Cholesterol oxidase 0.5 U/ml Catalase 80U/ml Magnesium chloride 10 mmol/l Second Reagents PIPES buffer, pH 7.0100 mmol/l 4-aminoantipyrine 4.0 mmol/l Peroxidase 2.4 U/ml Sodium azide0.1%  Emulgen B66 (HLB13.2) commercially 0.3%  available from KAOCORPORATION

To each of 4 samples having a volume of 4 μl containing purified HDL,LDL, VLDL and CM at a concentration of 100 mg/dl, respectively, 300 μlof the above-described first reagents which had been preliminarilywarmed at 37° C. were added and each of the resulting mixtures wasallowed to react at 37° C. for 5 minutes. Thereafter, 100 μl of thesecond reagents were added to each mixture and each of the resultantswas allowed to react for 5 minutes, followed by measurement ofabsorbance of each reaction mixture at 600 nm. Based on the measuredabsorbances, the amounts of cholesterol were calculated and the ratio ofthe thus calculated amount to the amount of the cholesterol in thesample was calculated, which is defined as capture ratio.

By this method, the hydrogen peroxide produced in the first step isdecomposed by catalase. On the other hand, the hydrogen peroxidegenerated in the second step forms a quinone pigment by reacting withHDAOS and 4-aminoantipyrine. The results are shown in Table 1.

TABLE 1 Capture Ratio CM VLDL LDL HDL <1.0% <1.0% <1.0% 86.6%

As shown in Table 1, by the above-described method, most of thecholesterol in HDL is quantified. On the other hand, the cholesterol inthe lipoproteins other than HDL is not substantially quantified. Thus,it can be seen that the cholesterol in HDL can be selectively quantifiedby the method of the present invention.

Example 2

Cholesterol in HDL in test samples was quantified by the same method asin Example 1 except that the test samples were sera of normalindividuals. On the other hand, the HDL cholesterol in the same testsamples was quantified by the precipitation method described in“Clinical Tests”, 23, 121 (1979). The correlation between the resultsobtained by these methods is shown in FIG. 1.

As shown in FIG. 1, the results of the quantification by these methodswell matched, so that it was proved that the cholesterol in HDL can beaccurately quantified by the method of the present invention.

Example 3

The same procedure as in Example 1 was repeated except that the firstand second reagents had the following compositions, respectively.

First Reagents HEPES buffer, pH 7.0 50 mmol/l DAOS 1.5 mmol/lCholesterol esterase 0.8 U/ml Cholesterol oxidase 0.5 U/ml Peroxidase1.0 U/ml Second Reagents HEPES buffer, pH 7.0 50 mmol/l4-aminoantipyrine 4.0 mmol/l Emulgen 911 (HLB13.7) commercially 0.3% available from KAO CORPORATION

By this method, the hydrogen peroxide produced in the first step reactswith DAOS by the action of peroxidase to form a colorless quinone. Onthe other hand, the hydrogen peroxide generated in the second stepreacts the remaining DAOS from the first step and with 4-aminoantipyrineadded in the second step by the action of peroxidase to form a quinonepigment. The results are shown in Table 2.

TABLE 2 Capture Ratio CM VLDL LDL HDL <1.0% <1.0% <1.0% 85.3%

As shown in Table 2, by the above-described method, most of thecholesterol in HDL is quantified. On the other hand, the cholesterol inthe lipoproteins other than HDL is not substantially quantified. Thus,it can be seen that the cholesterol in HDL can be selectively quantifiedby the method of the present invention.

Example 4

Cholesterol in HDL in test samples was quantified by the same method asin Example 3 except that the test samples were sera of normalindividuals. As in Example 2, the HDL cholesterol in the same testsamples was quantified by the precipitation method. The correlationbetween the results obtained by these methods is shown in FIG. 2.

As shown in FIG. 2, the results of the quantification by these methodswell matched, so that it was proved that the cholesterol in HDL can beaccurately quantified by the method of the present invention.

Example 5

First reagents and second reagents having the following compositions,respectively, were prepared.

First Reagents BES buffer, pH 7.0 100 mmol/l HDAOS 0.7 mmol/lCholesterol esterase 0.8 U/ml Cholesterol oxidase 0.5 U/ml Catalase 100U/ml Magnesium chloride 18 mmol/l Second Reagents BES buffer, pH 7.0 100mmol/l 4-aminoantipyrine 4.0 mmol/l Peroxidase 2.4 U/ml Sodium azide0.1%  Emulgen B66 (HLB13.2) commercially 1.17%  available from KAOCORPORATION Emulgen A90 (HLB14.5) commercially 0.13%  available from KAOCORPORATION

To each of 4 samples having a volume of 4 μl containing purified HDL,LDL, VLDL and CM at a concentration of 100 mg/dl, respectively, 300 μlof the above-described first reagents which had been preliminarilywarmed at 37° C. were added and each of the resulting mixtures wasallowed to react at 37° C. for 5 minutes. Thereafter, 100 μl of thesecond reagents were added to each mixture and each of the resultantswas allowed to react for 5 minutes, followed by measurement ofabsorbance of each reaction mixture at 600 nm. Based on the measuredabsorbances, the amounts of cholesterol were calculated and the ratio ofthe thus calculated amount to the amount of the cholesterol in thesample was calculated, which is defined as capture ratio.

By this method, the hydrogen peroxide produced in the first step isdecomposed by catalase. On the other hand, the hydrogen peroxidegenerated in the second step forms a quinone pigment by reacting withHDAOS and 4-aminoantipyrine. The results are shown in Table 3.

Capture Ratio CM VLDL LDL HDL <1.0% <1.0% <1.0% 98.0%

As shown in Table 3, by the above-described method, most of thecholesterol in HDL is quantified. On the other hand, the cholesterol inthe lipoproteins other than HDL is not substantially quantified. Thus,it can be seen that the cholesterol in HDL can be selectively quantifiedby the method of the present invention.

Example 6

Cholesterol in HDL in test samples was quantified by the same method asin Example 5 except that the test samples were sera of normalindividuals. On the other hand, the HDL cholesterol in the same testsamples was quantified by the precipitation method described in“Clinical Tests”, 23, 121 (1979). The correlation between the resultsobtained by these methods is shown in FIG. 3.

As shown in FIG. 3, the results of the quantification by these methodswell matched, so that it was proved that the cholesterol in HDL can beaccurately quantified by the method of the present invention.

1. A method for enzymatically quantifying cholesterol in high densitylipoprotein, comprising the steps of reacting a surfactant whichspecifically acts on high density lipoprotein with the high densitylipoprotein, and enzymatically quantifying cholesterol in said highdensity lipoprotein; wherein the surfactant substantially does not acton other lipoproteins.
 2. The method according to claim 1, wherein saidsurfactant which specifically acts on high density lipoprotein has ahydrophilicity lipophilicity balance (HLB) of 13-14.
 3. A method forenzymatically quantifying cholesterol in high density lipoprotein,comprising the steps of reacting a surfactant which specifically acts onhigh density lipoprotein with the high density lipoprotein, andenzymatically quantifying cholesterol in said high density lipoprotein;wherein said surfactant which specifically acts on high densitylipoprotein is a polyalkylene oxide derivative.
 4. The method accordingto claim 2, wherein said surfactant which specifically acts on highdensity lipoprotein is a polyalkylene oxide derivative.